Download LIVER GENE EXPRESSION DURING THE TRANSITION DURING THE DRY PERIOD

LIVER GENE EXPRESSION DURING THE TRANSITION
PERIOD IS ALTERED BY LEVEL OF ENERGY INTAKE
DURING THE DRY PERIOD
Juan J. Loor, Heather M. Dann, Nicole A. Janovick Guretzky, Robin E.
Everts, Rosane Oliveira, Cheryl A. Green, Noah B. Litherland, Sandra L.
Rodriguez-Zas, Harris A. Lewin, and James K. Drackley
TAKE HOME MESSAGES
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Microarray technology is a new molecular tool that allows simultaneous analysis of
thousands of genes in a tissue sample. We are the first research group in the world to
apply this advanced technology to problems of dairy cattle physiology and nutrition.
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Restricted energy intake before calving resulted in earlier (i.e. day -14 relative to
parturition) up-regulation of genes with key functions in liver fatty acid oxidation,
gluconeogenesis, and cholesterol synthesis, among others.
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Ad libitum feeding up-regulated a number of genes associated with liver triglyceride
synthesis and pro-inflammatory cytokines; these responses corresponded closely with
increased liver triglyceride concentrations early postpartum.
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Moderate overfeeding of energy in the dry period results in transcriptional changes
predisposing cows to fatty liver, which may compromise overall liver health during the
periparturient period.
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Restricted energy intake may confer an advantage to the cow by triggering adaptations of
molecular mechanisms in the liver well ahead of parturition.
INTRODUCTION
The DNA microarray “chip” is a new tool used to monitor the level of expression of thousands
of genes simultaneously by measuring the abundance of RNA in a tissue sample. Information
necessary for the synthesis of enzymes and proteins needed in biological processes by an
organism is contained in RNA. The messenger RNA (mRNA) thus serves as the “go-between”
that connects the information contained as DNA in the cell nucleus to production of enzymes or
proteins to carry out that information in the cell. By measuring the degree of up- or downregulation of a gene from an RNA sample of a cow’s tissue, we can begin the formidable task of
defining roles for each gene and understanding the interactions between sets of genes in a
particular tissue during different physiological states.
Suboptimal nutritional management during the transition period can impact the incidence of
clinical or subclinical disease and thus animal well-being. Many of the common periparturient
health disorders (e.g. fatty liver, ketosis) are strongly linked to energy balance. Increasing
evidence implicates subclinical ketosis and fatty liver as predisposing factors for energyassociated disease as well as other metabolic and infectious problems. Function of the adult liver
as well as the performance of essential functions in the body is controlled through the
coordinated expression of a large number of genes. Environmental signals, such as nutrition,
exert powerful control of liver gene expression.
Current practice is to increase energy density of pre-calving diets and to maximize feed intake
before parturition. Our recent data suggest that cows that are moderately overfed during the dry
period, even without becoming visually fatter, may be placed at greater risk for periparturient
health problems. A consistent finding in our studies and others is that cows allowed ad libitum
access to higher energy diets during the dry period have larger decreases in feed intake before
parturition, and lower feed intake post-partum. Therefore, lower energy diets may be beneficial
in maintaining more consistent feed intake around and after parturition.
Genomic technologies may help identify regulatory mechanisms in liver that are sensitive to
nutrient balance during the dry period. Here we report temporal expression profiling of more
than 6,300 unique genes in liver of dairy cows moderately overfed or restricted-fed a moderate
energy diet throughout the dry period using a cattle-specific, high-density cDNA. Evidence
indicates that moderate overfeeding of energy during the dry period results in transcriptional
changes predisposing cows to fatty liver and compromising overall liver health.
METHODS
Cows were selected from two groups of 12 cows enrolled in a large experiment to assess the
effects of ad libitum or restricted intake of moderate-energy diets during the entire dry period on
pre-partum metabolism and post-partum metabolism and performance. A corn silage-based diet
(26% of diet dry matter) providing 0.72 Mcal/lb during the far-off dry period (first 5 wk of an 8wk dry period) or providing 0.73 Mcal/lb during the close-up period (last 3 wk of the dry period)
was fed for ad libitum (160% of calculated energy requirements) or restricted (80% of
requirement) intake. After calving, all cows were fed a common lactation diet, balanced
according to NRC (2001) recommendations. Four multiparous Holstein cows were randomly
selected from each group. All cows underwent normal calving and were free from disorders.
Total RNA was extracted from liver tissue biopsies obtained on day -65 (dry-off), -30, -14, +1,
+14, +28, and +49 relative to parturition. The relative abundance of 7,872 mRNAs in each of the
samples was measured by DNA microarray developed at the University of Illinois. By using a
‘reference’ pool of mRNA collected from tissues not including liver, we were able to calculate
the abundance of those genes present in liver relative to their amount in the ‘reference’ sample.
RESULTS AND DISCUSSION
A total of 85 genes were ‘affected’ by the interaction of time and pre-partum diet, and 122 genes
were affected exclusively by pre-partum diet. Many of these genes are involved in key aspects
of lipid metabolism (e.g. fatty acid oxidation, triglyceride formation), carbohydrate metabolism
(e.g. synthesis of glucose or ‘gluconeogenesis’), cholesterol synthesis, and inflammatory
responses (e.g. proteins that activate the immune system). Some of the changes in liver gene
expression explain nutrition-induced differences in the capacity of liver to esterify or oxidize
long-chain fatty acids. For example, in cows fed ad libitum prepartum (AA; Fig. 1A), we
observed a linear increase in expression from day -30 to +14 relative to parturition of genes
associated with fat synthesis in liver such as 1-acylglycerol-3-phosphate-acyltransferase
(AGPAT1). In contrast, expression of genes with key functions in liver fatty acid oxidation such
as acyl-CoA dehydrogenase-very long-chain (ACADVL), carnitine palmitoyl transferase-1
(CPT1A), and adiponectin receptor-2 (ADIPOR2) was nearly 2-fold higher around parturition in
restricted-fed (RR, Fig. 1B) cows. These data show that restricted energy feeding prepartum
confers an advantage to the cow by triggering molecular adaptations in liver well ahead of
parturition, which are necessary to deal with greater influx and uptake of nonesterified fatty acids
(NEFA) by liver after calving. Taken together, these changes are consistent with greater
likelihood for fat accumulation in the liver after parturition in cows that had free access to
moderate-energy diets prepartum.
1.5
A
1.3
1.0
0.8
AA AGPAT1
RR AGPAT1
AA TNFAIP3
RR TNFAIP3
0.5
0.3
0.0
Fold change relative to day -65
-65
3.0
-30
-14
1
14
28
Day relative to calving
C
49
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
AA ADIPOR2
RR ADIPOR2
AA ACADVL
RR ACADVL
AA CPT1A
RR CPT1A
B
-65
-30
-14
1
14
28
49
Day relative to calving
AA PC
RR PC
2.5
2.0
1.5
1.0
0.5
0.0
-65
Fold change relative to day -65
Fold change relative to day -65
During the transition period, there was increased expression of a key gene (pyruvate carboxylase,
PC) involved in liver glucose synthesis in RR cows. Increased PC mRNA was likely associated
with the need to maintain blood glucose levels in RR cows; whereas in AA cows greater influx
of propionate (from greater grain intake), greater liver glucose output, and the resulting high
blood insulin levels could have decreased PC expression through a feedback mechanism. Lower
post-partum PC expression for AA cows implies that liver use of lactate and glucogenic amino
acids for glucose synthesis (i.e. gluconeogenesis) was reduced, which may have hindered the
ability of cows to cope with increasing glucose needs for milk production.
-30
-14
1
14
28
Day relative to calving
49
Fig. 1. Changes in mRNA abundance relative
to dry-off (day -65) for key lipid metabolism
(panels A and B) and glucose synthesis genes
(panel C). Cows were overfed (AA, ad
libitum) or slightly underfed (RR, restricted)
through the dry period but fed the same
lactation diet after calving. All comparisons
were statistically significant (P < 0.05).